Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana

Nitrate (NO3–) is an essential element and nutrient for plants and animals. Despite extensive studies on the regulation of nitrate uptake and downstream responses in various cells, our knowledge of the distribution of nitrogen forms in different root cell types and their cellular compartments is still limited. Previous physiological models have relied on in vitro biochemistry and metabolite level analysis, which limits the ability to differentiate between cell types and compartments. Here, to address this, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named nlsNitraMeter3.0, for the quantitative visualization of nitrate concentration and distribution at the cellular level in Arabidopsis thaliana. This biosensor was specifically designed for nitrate measurements, not nitrite. Through genetic engineering to create and select sensors using yeast, Xenopus oocyte, and Arabidopsis expression systems, we developed a reversible and highly specific nitrate sensor. This method, combined with fluorescence imaging systems such as confocal microscopy, allows for the understanding and monitoring of nitrate transporter activity in plant root cells in a minimally invasive manner. Furthermore, this approach enables the functional analysis of nitrate transporters and the measurement of nitrate distribution in plants, providing a valuable tool for plant biology research. In summary, we provide a protocol for sensor development and a biosensor that can be used to monitor nitrate levels in plants. Key features This protocol builds upon the concept of FRET biosensors for in vivo visualization of spatiotemporal nitrate levels at a cellular resolution. Nitrate levels can be quantified utilizing the biosensor in conjunction with either a plate reader or a fluorescence microscope.

n/a n/a Total n/a 500 mL a. Autoclave, 121 °C, 15 psi, 15 min b.For liquid medium, when hand-warm, add glucose from 40% sterile filtrated stock to a final concentration of 2% under a sterile hood (e.g., biosafety cabinet).c.For solid medium, add 20 g/L agar before autoclaving.Add sterile filtrated glucose from 40% stock to a final concentration of 2% when the medium is hand-warm before pouring plates.n/a to 1,000 mL Total n/a 1,000 mL a. Autoclave, 121 °C, 15 psi, 15 min b.For liquid medium, when hand-warm, add glucose from 40% sterile filtrated stock to a final concentration of 2% under a sterile hood (e.g., biosafety cabinet).c.For solid medium, add 20 g/L agar before autoclaving.Add sterile filtrated glucose from 40% stock to a final concentration of 2% when the medium is hand-warm before pouring plates.d.Adjust the pH of the -ura DropOut medium to pH 5.8 with NaOH before addition of agar and autoclaving.

A. FRET sensor design
For a detailed account of the generation of the sensor DNA constructs and the sensor mutants , please refer to Chen and Ho (2022).In this section, we just report a few concepts of the sensor design.

DNA constructs
Based on the FRET characteristic, we designed the Gateway expression clones with an insert of the bacterial (K.oxytoca) NasR/NIT domain (Figure 1).It takes approximately 10 min to read a full 96-well plate with the parameters mentioned above.For highly accurate analyses, measure only a few wells at a time to reduce differences in analysis time.It is also possible to use instruments with injectors that allow for immediate recording; use rapid switching between wells to record over time.
Note: The sensor exhibits functional activity when employed as a purified recombinant protein.(final concentration 100 μg/mL) and grow the culture at 28 °C for 16-24 h.f.Collect Agrobacterium cells by centrifugation at 3,000× g for 10 min at RT and discard the supernatant.
Then, gently resuspend cells in one volume of the freshly made dipping medium.g.Dilute Agrobacterium cells to 6 × 10 9 cells/mL.h.Spray the Agrobacterium on the floral part of the Arabidopsis.Then, lay down the dipped plants in a plastic basin and cover them with plastic wrap for 16-24 h to maintain high humidity.i.The next day, remove the cover and allow them to grow normally for one month in the greenhouse or the growth chamber; withhold watering when siliques turn brown.3. Select transformants on agar plates containing 1/2× MS medium with vitamins (PhytoTech Labs, M519) and kanamycin (30 mg/L).

D. Imaging the nitrate sensor in Arabidopsis with fluorescence microscopy confocal microscopy
Note: Although a fluorescence confocal microscope is the standard equipment used, light-sheet microscopy is another option.The settings for laser intensity, detector, and objective are similar to those for confocal microscopy.Please refer to the detailed procedure of the light-sheet system in the Supplementary information section.

Nitrate treatments on glass slides for confocal microscopy
a. Place seedlings on glass slides with 50 μL of solution, surround with a rectangle of vacuum grease, and cover with a square coverslip equal in height and half the width of the vacuum grease rectangle.ii.For other methods that can be used to obtain continuous images or video, please refer to the Supplementary information.

Data analysis
A. Fluorescence emission ratio response of purified NiMet3.0 to NO 3 − in vitro 1. Subtract background fluorescence of yeast (using cells transformed with vector only) from all fluorescence values (for both spectra as well as single point measurements).2. The solution addition might trigger a change in the energy transfer rate between the emission at 530 nm [Dx acceptor emission (DxAm)] and the emission at 488 nm [Dx donor emission (DxDm)] that could act as a FRET ratio change sensor (ΔDxAm/DxDm).Through several optimizations, we obtained a fusion construct that shows a significantly substrate-triggered positive ratio change (ΔDxAm/DxDm) (e.g., NiMet3.0)(Figure 2).Notes: a. NiMet3.0 expressed in yeast responds to nitrate addition by changing the fluorescence intensity of donor and acceptor emission (obtained with excitation at 428 nm).Aphrodite-t9 emission was unaffected and served as a control or reference for normalization (obtain ed at 500 nm excitation).Nitrate addition (5 mM) induced a decrease in the emission spectrum of the donor, and the emission of the acceptor increased (Figure 2A).Besides, since the Aphrodite-t9 emission is unaffected by nitrate when excited directly, Aphrodite-t9 emission can be used as a control and for normalization by using ratios instead of absolute values to compare between different cultures.b.The various nitrate concentrations from micromolar to millimolar were added externally to the primary root to monitor the NitraMeter sensor responses.The data showed that the FRET ratio changed to external nitrate addition was saturated after approximately 0.25-0.5 mM, indicating either that the NitraMeter sensor in root was all occupied by nitrate or the Vmax of NitraMeter sensor was reached after the concentrations of nitrate addition externally.

1 .
DNA constructs for expressing sensors in plants a. Insert open reading sequence of NasR or NasR-NR-R176A into the multiple cloning site of the p16-Kan vector (Jones et al., 2014): 5′-, a sequence coding for the SV40-derived nuclear localization signal LQPKKKRKVGG (Schuster et al., 2014); a sequence coding for Aphrodite; a Gateway cassette including attR1, Chloramphenicol resistance gene, ccdB terminator gene, and attR2; a sequence coding for mCerulean (mCer); and a sequence coding for the cMyc epitope tag -3′, or pZPFlip UBQ10-KAN vector under the control of the UBQ10 promoter.Note: The p16 promoter (Schuster et al., 2014) from the AT3G60245 gene encoding a 16S ribosomal subunit was used to drive the nuclear-localized NiMet3.0 fusion biosensor, whereas the CaMV 35S promoter (Battraw and Hall, 1990) was used to drive the NiMet3.0 and NiMet3.0-NR-R176Afusion biosensor in plants.b.Recombine in Gateway LR reactions with NasR or NasR-NR-R176A Entry Clones, resulting in NiMet3.0,NiMet3.0-NR-R176A, and nlsNiMet3.0expression clones.2. Generate transgenic plants using the Agrobacterium floral dip method a. Introduce sensors into Agrobacterium tumefaciens GV3101.b.Grow healthy Arabidopsis plants in 12 h of light, 50% humidity, and at 22 °C until they begin to bolt and produce floral inflorescences (3-4 weeks in a growth chamber).c.Remove siliques and mature flower clusters before floral dipping.d.Inoculate a single Agrobacterium colony that was transformed with sensors into 5 mL of liquid LB medium containing the appropriate antibiotics [spectinomycin (final concentration 100 μg/mL)] for binary vector selection.Incubate the culture at 28 °C overnight.e.The following morning, use this feeder culture to inoculate 200 mL of liquid LB with spectinomycin

9 Published:
Cite as: Chen, Y.N. and Ho, C.H. (2023).Fluorescent Biosensor Imaging of Nitrate in Arabidopsis thaliana.Bio-protocol 13(16): e4743.DOI: 10.21769/BioProtoc.4743.Aug 20, 2023 b.Exchange the nitrate treatment solution beneath the coverslip by addition to the left and removal from the right side of the coverslip.c.Acquire confocal images on a Zeiss 780 laser scanning microscope and use a 20×/0.8Plan -Apochromat dry objective or 40×/1.2C-Apochromat water objective.Excite CFP (440 nm) and yellow fluorescent protein (YFP; 514 nm) with lasers.Detect fluorescence emission using a GaAsP photomultiplier tube (PMT) detector, set to detect 463-508 nm for CFP, and a normal PMT detector, set to 520-585 nm for YFP.Set the laser power between 0.5% and 2% with detector gain set to 700-750 to image CFP or YFP.d.Acquire images at time points based on the purpose of the research (refer to the note below for details on time point settings).Acquire three-dimensional images, with a z-step size of 1.5 μm and half the diameter of the primary root axis in Arabidopsis.Notes: i.For example, if the purpose is to observe the nitrate distribution in the root after different concentrations of medium supplement, it is suitable to set the range and interval of the time points to less than an hour, unless the sample can be kept moist.Additionally, fluorescence blenching should be considered when continuously recording.

Figure 2 .
Figure 2. Fluorescence response of NiMet 3.0-and nlsNiMet 3.0-expressing yeast cells and Arabidopsis root.(A) Fluorescence emission wavelength scan (B) and emission ratio at 530 nm of purified NiMet3.0 protein with and without NO3 − .Nitrate concentration is indicated in the figures.Nitrate was able to trigger responses that were significantly different from the control (*, P < 0.0001, t-test).The presented data are mean ± SD of six biological repeats.(C) Three-dimensional images of nlsNiMet3.0emission ratios of 5-day-old root meristem zone in transgenic Col-0 before a NO3 − pulse, after the NO3 − pulse, and after removing the NO3 − .NO3 − (50 μM) was used.(D) Beeswarm and box plot of NO3 − concentrationdependent nlsNiMet3.0emission ratios for nuclei of root tips.****, P < 0.0001, Student's t-test.Means ± SD of three biological repeats are presented.